Project Summary Since the advent of imatinib, a specific kinase inhibitor used to treat chronic myeloid leukemia, further development of small molecule kinase inhibitors has been a promising avenue to combat cancer. In particular, cyclin-dependent kinase 2 (Cdk2) has been a promising anti-cancer target, as it is normally activated by cyclin E to proceed through the cell cycle at the G1/S phase transition, and hyperactivated in diseases such as ovarian, breast, and colorectal cancers. Historically, targeting Cdk2 involved designing molecules that bind its active site, leading to many off-target effects due to the homology of the ATP-binding site among kinases, especially other Cdk enzymes. However, our group recently discovered and characterized an allosteric binding pocket in Cdk2 bound to the dye 8-anilino-1-naphthalenesulfonic acid (ANS). There is no evidence that this pocket exists in other Cdk family members and therefore provides an outstanding opportunity to discover truly selective first-in-class Cdk2 inhibitors that could be devoid of off-target toxicity. By targeting the allosteric pocket, we hypothesize that novel inhibitors can be developed that will not bind to other Cdk family members given the non-homologous nature of this allosteric site. There is currently no known potent allosteric nor selective inhibitor of Cdk2. My proposal focuses on developing novel allosteric inhibitors using two approaches: chemical library development of ANS analogs (Aim 1) and discovery of new chemical matter that binds the allosteric pocket using high-throughput screening (HTS), fragment screening, virtual screening, and confirmation through biological testing (Aim 2). These techniques offer a multipronged approach to provide chemical and structural insight towards the development of a potent and selective allosteric inhibitor of Cdk2 to treat cancer. If successful, this array of strategies can serve as an example for kinase inhibitor design in general. This fellowship proposal will also provide training in medicinal chemistry, chemical biology, and translational science, contributing to a meaningful career as a physician scientist who appreciates all facets of the drug development process. This training will allow me to become an independent researcher who can bridge the gap between the scientists who discover new therapeutics and the physicians who test them. This work strongly aligns with the mission of NCI to incorporate novel treatments into clinical practice.